Physiological and Molecular Plant Pathology (2002) 61, 325±337
doi:10.1006/pmpp.2003.0445
Induction of an antioxidant enzyme system and other
oxidative stress markers associated with compatible
and incompatible interactions between chickpea
(Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceris
CA R M E N G A R C IÂ A - L I M O N E S 1, A N A H E RV AÂ S 2, J UA N A . N AVA S - COR T EÂ S 2,
R A FA E L M. J IM EÂ N E Z - D IÂ A Z 2,3 and M A N U E L T E N A 1*
1
Departamento de BioquõÂmica y BiologõÂa Molecular, ETSIAM, Universidad de CoÂrdoba, Apartado 3048, 14080 CoÂrdoba, Spain,
2
Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Cientõ®cas, Apartado 4084, 14080 CoÂrdoba, Spain and
3
Departamento de AgronomõÂa, ETSIAM, Universidad de CoÂrdoba, Apartado 3048, 14080 CoÂrdoba, Spain
(Accepted for publication 8 November 2002)
To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f.
sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine
oxidase (DAO), an apoplastic H2O2-forming oxidase, and several antioxidant enzymes, namely ascorbate
peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX)
and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of `WR315'
(resistant) and `JG62' (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the
pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel
electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by
Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD
activities, although such responses occurred earlier in the incompatible compared with the compatible
interactions. APX, GPX and GR activities were also increased in infected roots, but only in the
compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT,
SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible
one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and
did not reveal any di€erences in isoenzyme patterns between cultivars or between infected and noninfected plants. Further, Western blots revealed an increase in the root protein levels of APX in
the compatible interaction and in those of SOD in both compatible and incompatible interactions.
In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and
CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the
induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and
GPX (only in stems) activities are rather more associated with the establishment of the compatible
c 2003 Elsevier Science Ltd. All rights reserved.
*
interaction.
Keywords: Cicer arietinum; Fusarium oxysporum f. sp. ciceris; ascorbate peroxidase; catalase; diamine
oxidase; glutathione reductase; guaiacol peroxidase; superoxide dismutase; lipid peroxidation.
INTRODUCTION
The generation of active oxygen species (AOS), such as the
superoxide anion O2 and hydrogen peroxide (H2O2), is a
* Corresponding author. Tel.: ‡34-9572-18439; Fax: ‡349572-18563; E-mail address: [email protected] (M. Tena).
Abbreviations used in text: AOS, active oxygen species; APX,
ascorbate peroxidase; CAT, catalase; CMS, 1 : 9 : 2 corn mealsand-water mixture; DAO, diamine oxidase; DII, disease
intensity index; GPX, guaiacol peroxidase; GR, glutathione
reductase; MDA, malondialdehyde; NBT, nitroblue tetrazolium;
PDA, potato dextrose agar; PVPP, polyvinylpolypyrrolidone;
SAR, systemic acquired resistance; SOD, superoxide dismutase;
TBA, 2-thiobarbituric acid; VOPA, V8 juice-oxgall-PCNB agar.
0885-5765/03/$ - see front matter
common event associated with normal plant biochemical
processes including chloroplast and mitochondrial electron transport and oxidases in the plasma membrane. The
reactive nature of AOS makes them potentially harmful to
many cellular components. Thus, AOS accumulation
causes oxidative damage through actions such as lipid
peroxidation with membrane destruction, protein inactivation or DNA mutation. Fortunately, plants, similarly
to other aerobic organisms, are endowed with ecient
AOS-scavenging mechanisms which include both enzyme
and chemical antioxidant systems. These mechanisms are
expressed constitutively to cope with AOS formed under
normal conditions. However, they can also be induced
c 2003 Elsevier Science Ltd. All rights reserved.
*
326
C. GarcõÂa-Limones et al.
to maintain the lowest possible levels of AOS in
circumstances of enhanced production of such derivatives,
as usually occurs under diverse biotic and abiotic stresses.
Thus, both the formation of oxidized products, such as
lipid peroxidation products [2], and the induction of
antioxidant mechanisms, may be a sign of AOS overproduction and thereby of oxidative stress.
The transient production of AOS, in an oxidative burst,
is frequently an early plant response to pathogen attack
[6, 35, 40, 41, 52, 58]. AOS have been suggested to be
involved in plant defence responses in several ways:
(a) reinforcing plant cell-walls through cross-linking
reactions of lignin and proteins; (b) acting as toxic agents
against either the host plant cells, with development of
hypersensitive response (HR) and systemic acquired
resistance (SAR), or against the pathogens, killing them
or stopping their growth and development; and
(c) participating as second messengers in signalling routes
leading to the activation of plant defence-related genes.
H2O2 is the major AOS of the oxidative burst in plants,
since it is the most long-lived and able to cross plant cell
membranes and thereby act as a di€usible and relatively
lasting signal. AOS production, including O2 and H2O2,
has been especially well established in several plant tissue
[2, 3, 16±18, 53] and suspension-cultured cell systems
[4, 37, 38, 55] associated with the expression of an HR and
SAR. In contrast, very little is known about oxidative
metabolism in plant resistance reactions to pathogens that
do not induce HR, such as the necrotrophic fungi that
invade the plant vascular system.
Chickpea (Cicer arietinum L.) is one of the most important
food legumes grown worldwide, especially in dry areas of
the Indian subcontinent [47]. In the European Union,
chickpea production is concentrated mainly in the
Mediterranean Basin, with Spain being the principal
producer. Fusarium wilt, caused by Fusarium oxysporum
Schlechtend.: Fr. f. sp. ciceris (Padwick) Matuo and
K. Sato, is a major constraint to chickpea production
worldwide [29]. Annual chickpea yield losses from
fusarium wilt vary from 10 to 15 % [29, 54], but the
disease can completely destroy the crop under unfavourable conditions [24]. Histological analyses of chickpeaF. oxysporum f. sp. ciceris interactions have not revealed any
symptom of localized rapid cell death in infected roots of
resistant cultivars [51], thus denoting that in this system
the resistance reaction is apparently not associated with a
hypersensitive response. The aim of the present work was
to investigate the possible role of AOS production in
fusarium wilt of chickpea. To this end, we have studied the
rate of lipid peroxidation and the levels of various
antioxidant enzymes (namely ascorbate peroxidase
[APX, EC 1.11.1.11], catalase [CAT, EC 1.11.1.6],
guaiacol-dependent peroxidase [GPX, EC 1.11.1.7],
glutathione reductase [GR, EC 1.6.4.2], and superoxide
dismutase [SOD, EC 1.15.1.1]), as well as those of diamine
oxidase (DAO, EC 1.4.3.6), an apoplastic H2O2-forming
oxidase that is particularly active in leguminous plants
[20], in roots and stems of resistant and susceptible
chickpeas infected with the highly virulent race 5 of
F. oxysporum f. sp. ciceris.
MATERIALS AND METHODS
Plant and fungal material
Two `desi' chickpeas (small, ridged, brown seed), `JG62'
and `WR315', which are susceptible and resistant,
respectively, to race 5 of F. oxysporum f. sp. ciceris (Foc),
were used throughout the study. Seeds, selected on the
basis of size and colour uniformity and absence of spots
and other signs of injury in their coats, were surfacesterilised in 2 % NaOCl for 3 min, washed three times in
sterile distilled water, and germinated on autoclaved
layers of paper towels in moist chambers at 288C, in
darkness for 48 h.
Isolate Foc 8012 of F. oxysporum f. sp. ciceris race 5 (Foc 5)
[32] was used in this study. This isolate was obtained from
infected chickpeas in Southern Spain and causes vascular
wilt in susceptible chickpea cultivars [54]. Fresh cultures of
Foc 8012 were obtained from a monoconidial culture
stored on sterile sand in test tubes at 48C. A few infested
sand grains from the stock culture were placed on potatodextrose agar (PDA) in Petri dishes and incubated at 258C
and a 12 h photoperiod of ¯uorescent and near-u.v. light
at 36 mE m 2 s 1 for 7 days. Inoculum was then increased
on a 1 : 9 : 2 corn meal-sand-water mixture (CMS).
Aliquots of 0.4 kg CMS placed in 1 liter-¯asks were each
infested with 16 disks (0.5 cm2 in surface) of PDA cut from
the growing edge of the fungal cultures. Similar aliquots
bearing disks of sterile PDA served as controls. Cultures in
¯asks were incubated for 14 days under the same
conditions as PDA cultures. Flasks were hand-agitated
vigorously every 2 days to facilitate homogeneous fungal
colonization of the substrate. Inoculum density was
estimated by dilution plating onto V8 juice-oxgall-PCNB
agar (VOPA), a Fusarium selective medium [9]. From 1 g
infested CMS, serial dilutions were prepared in 0.1 %
water agar and the 10 6 to 10 8 dilutions were plated onto
VOPA. Cultures were incubated at conditions described
above for 5 days, and the number of colonies were counted.
Plant growth and inoculation, sampling and evaluation of
disease progress
Germinated chickpea seeds, selected for uniformity
(length of radicleˆ1±2 cm), were sown into earthen pots
(15 cm diameter, 0.6 l capacity) ®lled with an autoclaved
soil mixture (clay loam : peat, 2 : 1, v/v) (controls), or with
the same soil mixture amended with infested CMS at
approximately 400 000 cfu g 1 soil [45]. The experiment
Induction of an antioxidant enzyme system and other oxidative stress markers
consisted of four treatments ( JG62-Foc 5, WR315-Foc 5,
and their respective non-infested controls). There were 15
pots per treatment and ®ve seeds per pot. Plants were
grown in a growth chamber at 258C, 60±90 % relative
humidity, and a 14 h photoperiod of ¯uorescent light at
360 mE m 2 s 1. Plants were observed daily for development of symptoms, watered as needed, and fertilised
weekly with 100 ml per pot of Hoagland's nutrient
solution [27]. These growth conditions are optimal for
development of chickpea fusarium wilt [36]. The experiment was repeated three times.
Chickpea root and stem samples of inoculated and
control plants were collected at three characteristic time
points after inoculation: (1) before symptom development
(10 d after inoculation); (2) at the time of appearance of
the ®rst disease symptoms, e.g. initial ¯accidity of lea¯ets
followed by a dull-green discoloration (between 15 and
17 d after inoculation); and (3) when all plants had
developed disease symptoms, e.g. severe leaf chlorosis,
¯accidity, and wilt (between 20 and 22 d after inoculation). At each sampling date, plants in ®ve pots per
treatment were collected. Plants were carefully removed
from the pots in order to cause minimal root injury, washed
free of soil under tap water and then in distilled water, and
separated into root and shoot portions. Stems free of leaves
were divided into lower and upper halves. Both roots and
lower or basal stem portions were collected for biochemical
analyses. These tissues were frozen in liquid nitrogen and
processed immediately for enzyme extraction (see below).
Development of disease was assessed by the incidence and
severity of symptoms. Severity symptoms on individual
plants were rated on a scale from 0 to 4 according to
percentage of foliage with chlorosis or necrosis in acropetal
progression: 0 ˆ 0 %, 1 ˆ 1±33 %, 2 ˆ 34±66 %, 3 ˆ 67±
100 %, and 4 ˆ dead plant [25, 26]. The incidence and
severity data within a pot were used to calculate a disease
intensity index (DII) as described elsewhere [25]. At each
sampling date, isolations were made from stem segments of
sampled plants to determine the occurrence of vascular
infections. To this end, ®ve stems from each treatment were
selected at random, divided into three segments (T1 to T3
from the apex to the base), surface-desinfected in 1 %
NaOCl for 2 min, cut into 5 mm long pieces, and plated
onto VOPA. Plates were incubated as described above for
fungal cultures.
Enzyme extraction and activity assays
Frozen root and stem samples were crushed to a ®ne
powder in a mortar under liquid nitrogen. Soluble
proteins were extracted by resuspending the powder in
four volumes of 50 mM potassium phosphate bu€er,
pH 7.5, containing 1 mM EDTA, 1 mM PMSF, 5 mM
sodium ascorbate and 5 % (w/v) PVPP. The homogenate was strained through two layers of Miracloth
327
(Calbiochem) and centrifuged at 17 000 g for 10 min.
The supernatant was divided into aliquots, frozen in
liquid nitrogen and stored at 808C for further analysis.
All above operations were carried out at 0±48C.
Levels of the various antioxidant enzyme and DAO
activities in plant extracts were measured spectrophotometrically. APX, CAT, GPX and GR were assayed at
258C in a ®nal reaction volume of 0.6 ml. APX activity
was determined according to [44] with minor modi®cations. The reaction mixture consisted of 50 mM potassium phosphate bu€er, pH 7.0, 0.25 mM sodium ascorbate,
5 mM H2O2 and 50 ml of enzyme extract. The reaction was
started by adding H2O2 and the oxidation of ascorbate was
determined by the decrease in A290 (e ˆ 2.8 mM 1 cm 1).
One unit of APX activity is de®ned as the amount of
enzyme that oxidizes 1 mmol min 1 ascorbate under the
above assay conditions.
CAT activity was assayed according to [8] with minor
modi®cations. The reaction medium consisted of 50 mM
potassium phosphate bu€er, pH 7.0, 20 mM H2O2 and
between 10 and 30 ml of enzyme extract. The reaction
was started by adding H2O2 and the decrease in A240
(e ˆ 36 mM 1 cm 1), produced by H2O2 breakdown was
recorded. One CAT unit is de®ned as the amount of
enzyme necessary to decompose 1 mmol min 1 H2O2
under the above assay conditions.
GPX activity was assayed by a modi®cation of the
method described in [50]. The reaction mixture consisted
of 100 mM potassium phosphate bu€er, pH 6.5, 15 mM
guaiacol, 0.05 % (v/v) H2O2 and 60 ml enzyme extract
diluted between 1 : 40 and 1 : 80 (v/v) with assay bu€er.
The reaction was started by adding H2O2 and the
oxidation of guaiacol was determined by the increase in
A470 (e ˆ 26.6 mM 1 cm 1). One GPX unit is de®ned as
the amount of enzyme that produces 1 mmol min 1
oxidized guaiacol under the above assay conditions.
GR activity was assayed according to the method
described in [48] with minor modi®cations. The assay
mixture consisted of 50 mM potassium phosphate bu€er,
pH 7.5, 3.5 mM MgCl2, 0.15 mM NADPH, 0.5 mM
oxidized glutathione and 180 ml of enzyme extract. The
reaction was started by adding NADPH and oxidation of
this compound was determined by the decrease in A340
(e ˆ 6.2 mM 1 cm 1). One GR unit is de®ned as the
amount of enzyme that oxidizes 1 mmol min 1 NADPH
under the above assay conditions.
SOD activity was determined from the inhibition of the
photochemical reduction of nitroblue tetrazolium (NBT)
in the presence of ribo¯avin, according to [22]. The
reaction mixture (1.5 ml) consisted of 50 mM potassium
phosphate bu€er, pH 7.8, 0.1 mM EDTA, 13 mM
methionine, 75 mM NBT, 2 mM ribo¯avin and di€erent
volumes, between 10 and 100 ml, of enzyme extract. The
reaction was started by adding ribo¯avin and A560 was
measured after 12 min incubation at room temperature
328
C. GarcõÂa-Limones et al.
under continuous light. One SOD unit was de®ned as the
amount of enzyme (volume of enzyme extract) that
inhibits the rate of NBT reduction by 50 % under the
above assay conditions.
DAO activity was assayed according to [28] with
minor modi®cations. The reaction mixture (1.22 ml)
consisted of 100 mM potassium phosphate bu€er, pH 7.0,
10 mM putrescine, 0.16 mg ml 1 o-aminebenzaldehyde
and between 25 and 50 ml of enzyme extract. The mixture
was incubated at 378C with continuous agitation for
30 min. The reaction was stopped by adding 200 ml 10 %
(w/v) trichloroacetic acid then the reaction mixture was
centrifuged in a Microfuge for 15 min at top velocity and
the A435 of the supernatant was determined. One DAO
unit is de®ned as the amount of enzyme that oxidizes
1 mmol min 1 substrate (e ˆ 1.9 mM 1 cm 1) under the
above assay conditions.
In all assays the blank consisted of the components of
the reaction mixture except for the enzyme extract, which
was replaced by an equal volume of the assay bu€er.
In the case of the GR assay, an additional blank without
oxidized glutathione was included in order to account for
the presence in the extracts of other enzyme activities able
to oxidize NADPH. In the SOD assay, the enzyme blank
was taken as 100 % rate of NBT photochemical
reduction. In the remaining cases the enzyme blanks
were subtracted from the assay measurements.
Protein in enzyme extracts was determined by the
Bradford method [11] with BSA as a standard.
Native PAGE and enzyme activity staining
Electrophoretic separation of APX, CAT, GPX and
SOD isoenzymes was performed by native PAGE
[15] using a Mini-Protean II electrophoresis system
(Bio-Rad Laboratories). Electrophoresis was performed
at 48C for 45±50 min and a constant voltage of 200 V
using a 25 mM Tris, 192 mM glycine solution, pH 8.3 as
running bu€er. As an exception, the APX electrophoretic
separation was made at 100 V for 3 h. For CAT and
GPX, 3 % stacking and 5 % resolving polyacrylamide gels
were used, whereas for APX and SOD, 4 % and 10 %
stacking and resolving gels, respectively, were used.
Samples were applied in 62.5 mM Tris±HCl bu€er, pH
6.8, containing 10 % (v/v) glycerol and 0.025 % (w/v)
bromophenol blue. Equal amounts of protein, 18±20 mg
for root samples and 30±40 mg for stem samples, were
loaded and after electrophoresis gels were stained for
enzyme activities according to well established protocols,
as indicated.
APX was stained according to the method described in
[43], which is based on the inhibition of NBT reduction
by ascorbate. Following electrophoretic separation, gels
were equilibrated with 50 mM potassium phosphate
bu€er, pH 7.0, containing 2 mM sodium ascorbate for
30 min (the bu€er was changed each 10 min). Thereafter, gels were incubated in the above bu€er amended
with 4 mM sodium ascorbate and 2 mM H2O2 for 20 min.
Gels were then washed in the phosphate bu€er alone
for 1 min, stained in 50 mM potassium phosphate bu€er,
pH 7.8, amended with 28 mM TEMED and 2 mM NBT,
and agitated gently for 2±3 min up to appearance of
clear bands on an intense blue background due to NBT
reduction by ascorbate.
CAT was revealed according to [13]. Gels were washed
in distilled water and incubated in 50 mM potassium
phosphate bu€er, pH 7.0, containing horseradish peroxidase (50 mg ml 1) for 45 min in darkness. Then, H2O2 up
to 5 mM concentration was added to the incubation
mixture and incubation resumed for 10 min. Gels were
then washed twice in distilled water and stained in the
above phosphate amended with 0.5 mg ml 1 3,30 -diaminobenzidine (DAB) for 3±4 min. CAT activity appeared
as clear bands on a brown±orange background due to
DAB oxidation.
GPX was stained according to the method described in
[50]. Gels were equilibrated with 100 mM potassium
phosphate bu€er, pH 6.5, for 15 min, then incubated in a
12.5 mM guaiacol solution containing 1.7 mM benzidine
and 12 mM H2O2 up to appearance of brown±orange
bands against a clear background.
SOD activity was localized on gels by the method
described in [7]. This method is based on NBT reduction
by photochemically generated superoxide from ribo¯avin
and TEMED, and the inhibition of that reaction by the
enzymatic breakdown of O2 . Gels were incubated in
50 mM potassium phosphate bu€er, pH 7.8, amended
with 2.45 mM NBT, in the dark for 20 min. Subsequently, the above solution was replaced by the same
bu€er containing 28 mM ribo¯avin and 28 mM TEMED,
and incubation resumed for 15 min in darkness. Then,
gels were submerged in the above phosphate bu€er alone
and illuminated with white light until maximal contrast
between clear SOD bands against a blue background
(about 3±5 min) was attained. In order to identify
di€erent SOD forms, gels were preincubated with
selective inhibitors (2 mM KCN for inhibiting CuZnSODs and 5 mM H2O2 for inhibiting both CuZn- and
Fe-SODs) as described in [46].
SDS±PAGE and Western blot analysis
Enzyme extracts from roots were subjected to SDS±
PAGE and immunoblotting. The extracts were added to
an equal volume of 125 mM Tris±HCl bu€er, pH 6.8,
containing 4 % (w/v) SDS, 20 % (v/v) glycerol and
20 mM DTT, and the mixtures were heated at 958C
for 5 min. SDS±PAGE of denatured samples was
performed on 3 % stacking/12 % resolving slab gels in a
Mini-Protean II electrophoresis system, according to
Induction of an antioxidant enzyme system and other oxidative stress markers
the method of Laemmli [34]. For Western blot analysis,
proteins were transferred to polyvinylidene di¯uoride
membranes (Immobilon P, Millipore) in 10 mM 3-(cyclohexylamine)-1-propanesulphonic acid bu€er, pH 11.0,
containing 10 % (v/v) methanol, using a semi-dry
electrobot apparatus (Bio-Rad Laboratories) at
1.5 mA cm 2 for 2.5 h. Blots were incubated with
1 : 500 diluted antisera against cucumber APX [14],
Equisetum CuZn-SOD [33], or pea Mn-SOD. The
respective protein bands were visualized with horseradish
peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad
Laboratories, 1 : 10 000 dilution) according to the chemiluminiscence method described in [39].
329
Similarly, about 30 % of such plants were colonized
by Foc 5 in their middle and uppermost stem sections
(T2 and T1 stem samples, respectively). At the end of
disease development (between 20 and 22 days after
inoculation), DII reached near to 50 % and more than
90 % of plants showed vascular infection at T3 and about
60 and 20 % of these latter plants were infected at T2 and
T1, respectively. For the resistant cv. WR315 isolations
showed no evidence of systemic infection in stems of
inoculated plants at any sampling time.
Lipid peroxidation in root and stem samples
The level of lipid peroxidation products in root and stem
extracts were determined as 2-thiobarbituric acid (TBA)
reactive substances, which are mainly malondialdehyde
(MDA), by measuring the increase in A535 due to
formation of the TBA-MDA complex [12]. In these
assays, 0.2 ml of crude extracts were thoroughly mixed
with 0.4 ml of TBA reagent (a solution containing 15 %
w/v trichloroacetic acid and 0.375 % w/v TBA in 0.25 N
hydrochloric acid). The mixtures were heated for 15 min
at 1008C, cooled and cleared by centrifugation at 1000 g
for 10 min. Results were expressed as A535 per gram of
plant fresh weight.
The MDA levels in `WR315'- and `JG62'-inoculated
plants as well as those in their respective healthy controls
are shown in Fig. 1. In root samples [Fig. 1(A)], infection
by the pathogen resulted in a transient increase in
the MDA content in both cultivars compared with the
control; however, such an increase was higher (56 %,
P 5 0.05) and occurred earlier (sampling date 1) in
the resistant `WR315' compared with the susceptible
`JG62' (22 % increase, P 5 0.05, at sampling date 2).
In stem samples [Fig. 1(B)], the only signi®cant increase
in MDA levels resulting from infection by the pathogen
occurred in `JG62' (16 %, P 5 0.05) at sampling date 3.
In inoculated `WR315', there was a very little, not
signi®cant increase in the MDA content at sampling
date 1.
Analysis of data
Antioxidant enzymes and DAO activities
Spectrophotometric enzyme activity and lipid peroxidation data are presented as means of three readings from
three independent experiments. The signi®cant di€erences between means were determined by Student's t test.
Di€erences were considered to be signi®cant at P 5 0.05.
Enzyme activity staining after PAGE and immunoblotting after SDS±PAGE were repeated at least three or two
times, respectively. Photographs from one representative
experiment are presented here.
In roots of healthy, non-inoculated control plants, CAT
activity was higher in the susceptible (JG62) than in the
resistant (WR315) cv. [Fig. 2(B)], whereas the reverse
held true for GR activity [Fig. 2(D)]. Activity levels for
APX [Fig. 2(A)], GPX [Fig. 2(C)], SOD [Fig. 2(E)]
and DAO [Fig. 2(F)] were somewhat similar in both
cultivars.
Enzyme activities were in¯uenced by Foc 5 infection;
however, there were quantitative or time course di€erences in some of the observed changes when the
compatible (i.e. involving the susceptible `JG62') and
the incompatible (i.e. involving the resistant `WR315')
interactions were compared. CAT [Fig. 2(B)] and SOD
[Fig. 2(E)] activities in roots were increased by infection
in both interactions, although the induction response
occurred earlier in `WR315' than in `JG62'. Thus, for
CAT activity the maximal increase with respect to noninoculated control (110 %, P 5 0.05) was produced at
sampling date 1 in `WR315' but it was delayed to date 2
in `JG62' (87 %, P 5 0.01) [Fig. 2(B)]. Similarly, the
maximal increases in SOD activity with respect to noninoculated controls [Fig. 2(E)] occurred at sampling date
2 for `WR315' (46 %, P 5 0.05) and at date 3 for `JG62'
(30 %, P 5 0.05). Conversely, APX and GR activities
Lipid peroxidation products
RESULTS
Disease development in the susceptible cv. JG62
Ten days after inoculation plants were symptomless as
indicated by a very low DII value, i.e. less than 1 %;
however, isolations showed that about 30 % of such
plants were systemically infected in their lower stem
sections (T3 stem samples). Between 15 and 17 days after
inoculation, plants had developed the ®rst symptoms
characteristic of vascular wilt, with DII values near to
10 %. In this case, isolations showed that about 50 % of
plants were systemically infected at level of T3 samples.
330
C. GarcõÂa-Limones et al.
F I G . 1. Levels of malondialdehyde (MDA) in roots (A) and stems (B) of chickpea cvs. JG62 and WR315 infected by the wiltinducing race 5 of F. oxysporum f. sp. ciceris. MDA contents were analysed at three times, corresponding to seedlings without
symptoms (sampling date 1), at the onset of the ®rst symptoms (sampling date 2), and when all plants had developed wilt symptoms
(sampling date 3). (q) and (D) correspond to non-infected (control) and Foc 5-infected plants of the susceptible cv. JG62,
respectively, whereas (K) and (Q) correspond to non-infected (control) and Foc 5-infected plants of the resistant cv. WR315,
respectively. Data are means + SD of triplicate samples from three independent experiments. * indicates means that di€er
signi®cantly from control at P 5 0.05.
in roots were only signi®cantly induced in the compatible
interaction. As shown in Fig. 2(A), infection by Foc 5
remarkably increased APX activity in roots of `JG62',
where it reached its maximum level with respect to
control at sampling date 3 (235 %, P 5 0.01). At
sampling date 3, there was also an increase in APX
activity in roots of the resistant cv. WR315, but that was
not signi®cant [Fig. 2(A)]. GR activity was signi®cantly
increased in roots of inoculated JG62 at sampling dates 1
and 2, reaching the greatest induced value with respect to
control at date 2 (286 %, P 5 0.01). However, this
activity was little a€ected, if any, in similarly infected
roots of the resistant cv. WR-315 [Fig. 2(D)]. Finally,
there was no signi®cant change in GPX and DAO
F I G . 2. Levels of APX (A), CAT (B), GPX (C), GR (D), SOD (E), and DAO (F) activities in non-infected and Foc 5-infected roots
of chickpea cvs. JG62 and WR315. Activities were analysed at three times, corresponding to seedlings without symptoms (sampling
date 1), at the onset of the ®rst symptoms (sampling date 2), and when all plants had developed wilt symptoms (sampling date 3).
(q) and (D) correspond to non-infected (control) and Foc 5-infected plants of the susceptible cv. JG62, respectively, whereas (K)
and (Q) correspond to non-infected (control) and Foc 5-infected plants of the resistant cv. WR315, respectively. Data are
means + SD of triplicate samples from three independent experiments. *,** indicate means that di€er signi®cantly from control at
P 5 0.05 and P 5 0.01, respectively.
Induction of an antioxidant enzyme system and other oxidative stress markers
activities in roots infected by Foc 5 in either `JG62' or
`WR315'. However, GPX activity showed an increasing
trend in infected versus non-infected roots in successive
post-inoculation periods, especially in the compatible
interaction [Fig. 2(C)]. Conversely, DAO activity
showed a declining trend in infected versus non-infected
plants, which was more pronounced in `JG62' than in
`WR315' [Fig. 2(F)].
The levels of antioxidant enzyme and DAO activities
were also determined in stem samples of non-infected and
infected plants (Fig. 3). With respect to non-infected
plants, some quantitative di€erences in enzyme activities
between stem and root tissues were found. Thus, CAT
and GR speci®c activities were clearly higher in stems
[Fig. 3(B) and (D)] than in roots [Fig. 2(B) and (D)],
whereas the reverse held true for the remaining enzymes,
especially for GPX where the speci®c activity in roots was
about tenfold that in stems [Figs 2(C) and 3(C)]. In spite
of these quantitative di€erences in activity between
enzymes from root and stem tissues, both enzyme sets
showed relatively similar variations in activity levels when
the two cultivars were compared. Thus, similarly to that
found in roots, levels of GR activity in stems of `WR315'
were higher than those in `JG62' whereas levels of APX,
GPX, SOD and DAO activities were similar in both
331
cultivars (Fig. 3). As an unique exception to above, the
levels of CAT activity in stems of `WR315' were similar
to, or even higher than, those in `JG62' [Fig. 3(B)].
With respect to infected plants, the only signi®cant
increase of antioxidant enzyme activities in stems
occurred in the compatible `JG62'±Foc 5 interaction
(Fig. 3). Thus, in `JG62' stems APX [Fig. 3(A)], CAT
[Fig. 3(B)], GPX [Fig. 3(C)] and SOD [Fig. 3(E)]
activities were signi®cantly induced during disease
development. For CAT and SOD activities, higher levels
occurred in infected compared with non-infected control
plants at all sampling dates, although the maximal
inductions were obtained at sampling dates 2 (57 %,
P 5 0.05) and 3 (29 %, P 5 0.05), respectively. For APX
and GPX, in turn, only the activity levels at sampling
date 3 were signi®cantly higher (74 %, P 5 0.05, and
140 %, P 5 0.05, respectively) in infected than in noninfected plants. The remaining antioxidant enzyme, GR,
also increased in stems of `JG62'-infected plants, although
the di€erences with non-infected controls were not
signi®cant [Fig. 3(D)]. On the contrary, none of the
above antioxidant enzymes were signi®cantly increased,
not even exhibited any increasing trend, in stems of
infected `WR315' as compared with non-infected controls
[Fig. 3(A)±(E)]. In contrast with results for antioxidant
F I G . 3. Levels of APX (A), CAT (B), GPX (C), GR (D), SOD (E), and DAO (F) activities in non-infected and Foc 5-infected
stems of chickpea cvs. JG62 and WR315. Activities were analysed at three times, corresponding to seedlings without symptoms
(sampling date 1), at the onset of the ®rst symptoms (sampling date 2), and when all plants had developed wilt symptoms (sampling
date 3). (q) and (D) correspond to non-infected (control) and Foc 5-infected plants of the susceptible cv. JG62, respectively,
whereas (K) and (Q) correspond to non-infected (control) and Foc 5-infected plants of the resistant cv. WR315, respectively. Data
are means + SD of triplicate samples from three independent experiments. *,** indicate means that di€er signi®cantly from control
at P 5 0.05 and P 5 0.01, respectively.
332
C. GarcõÂa-Limones et al.
enzymes, DAO in stems increased signi®cantly in the
incompatible `WR315'±Foc 5 interaction at sampling
date 1 (55 %, P 5 0.05), whereas no enhancement in this
activity was evidenced in the compatible interaction at
any of sampling dates [Fig. 3(F)].
Electrophoretic analysis of antioxidant enzyme activities
The activities of the enzymes directly involved in AOS
scavenging, namely APX, CAT, GPX and SOD, were
analysed by native-PAGE in the same root and stem
extracts used for the above described spectrophotometric
assays. Results (Figs 4±7) did not reveal any di€erences
in the respective isoenzyme patterns of such enzyme
activities either between cultivars or between infected
and non-infected plants.
APX showed ample multiplicity in roots and stems.
However, one form of similar intermediate mobility,
denoted as APX III and V in the electrophoregrams of
root and stem extracts, respectively, was highly predominant in both organs (Fig. 4). This most abundant APX
isoform increased in both roots and stems of `JG62' at date
3 as a result of Foc 5 infection [Fig. 4(A) and (B), third
panel, lane 2 versus 1]. This would be in good accordance
with the signi®cantly increased APX activity levels found
in these samples [Figs 2(A) and 3(A)]. Some increase in
band intensity was also observed in `JG62'-infected plants
F I G . 4. Native PAGE analysis for APX activity from noninfected and Foc 5-infected roots (A) and stems (B) of chickpea
cvs. JG62 and WR315. Activity was analysed at three times,
corresponding to seedlings without symptoms (sampling date 1,
left panels), at the onset of the ®rst symptoms (sampling date 2,
central panels) and when all plants had developed wilt
symptoms (sampling date 3, right panels). 1 and 2 are root
and stems samples from non-infected and Foc 5-infected `JG62'
plants. 3 and 4 are root and stems samples from non-infected
and Foc 5-infected `WR315' plants, respectively. In all cases,
the same quantity of protein was loaded, 18 mg for roots samples
(A) and 33 mg for stems samples (B).
F I G . 5. Native PAGE analysis for CAT activity from noninfected and Foc 5-infected roots (A) and stems (B) of chickpea
cvs. JG62 and WR315. Activity was analysed at three times,
corresponding to seedlings without symptoms (sampling date 1,
left panels), at the onset of the ®rst symptoms (sampling date 2,
central panels) and when all plants had developed wilt
symptoms (sampling date 3, right panels). 1 and 2 are root
and stems samples from non-infected and Foc 5-infected `JG62'
plants. 3 and 4 are root and stems samples from non-infected
and Foc 5-infected `WR315' plants, respectively. In all cases,
the same quantity of protein was loaded, 18 mg for roots samples
(A) and 33 mg for stems samples (B).
F I G . 6. Native PAGE analysis for GPX activity from noninfected and Foc 5-infected roots (A) and stems (B) of chickpea
cvs. JG62 and WR315. Activity was analysed at three times,
corresponding to seedlings without symptoms (sampling date 1,
left panels), at the onset of the ®rst symptoms (sampling date 2,
central panels) and when all plants had developed wilt
symptoms (sampling date 3, right panels). 1 and 2 are root
and stems samples from non-infected and Foc 5-infected `JG62'
plants. 3 and 4 are root and stems samples from non-infected
and Foc 5-infected `WR315' plants, respectively. In all cases,
the same quantity of protein was loaded, 18 mg for roots samples
(A) and 33 mg for stems samples (B).
Induction of an antioxidant enzyme system and other oxidative stress markers
333
F I G . 7. Native PAGE analysis for SOD activity from non-infected and Foc 5-infected roots (A) and stems (B) of chickpea cvs. JG62
and WR315. Activity was analysed at three times, corresponding to seedlings without symptoms (sampling date 1, left panels), at
the onset of the ®rst symptoms (sampling date 2, central panels) and when all plants had developed wilt symptoms (sampling date
3, right panels). 1 and 2 are root and stems samples from non-infected and Foc 5-infected `JG62' plants. 3 and 4 are root and stems
samples from non-infected and Foc 5-infected `WR315' plants, respectively. In all cases, the same quantity of protein was loaded,
18 mg for roots samples (A) and 33 mg for stems samples (B).
for the two less mobile root isoforms APX I and II at
sampling dates 2 and 3 [ Fig. 4(A), second and third
panel, lane 2 versus 1]. Conversely, in `WR315' the
increases in intensity of the di€erent APX bands in
infected root and stem samples were very scanty or null
compared with non-inoculated controls (Fig. 4).
CAT electrophoregrams revealed the presence of an
unique isoform in both root and stem extracts (Fig. 5).
In root extracts, the intensity of the CAT band in the
di€erent samples [Fig. 5(A)] was in general well
correlated with the enzyme activity level previously
assayed in such samples [Fig. 2(B)]. In stem samples,
an increased CAT band occurred in `JG62'-infected
plants compared with non-infected controls, mainly at
sampling date 3, but not in `WR315'-infected plants
[Fig. 5(B)]. This was again in good accordance with the
spectrophotometrically assayed levels of this enzyme
activity [Fig. 3(B)].
For GPX, the electrophoregrams indicated that two
isoforms occurred in roots, a highly predominant form of
very low mobility and a minor form, whereas only the low
mobility band was evident in stems (Fig. 6). Whereas
infection by Foc 5 did not induce GPX band intensities in
root samples of either of the two cultivars [Fig. 6(A)],
stem samples from infected plants showed, as compared
with their respective non-infected controls, increases in
GPX band intensity. Such increases occurred for
`WR315' at sampling date 2 and for `JG62' at sampling
date 2, but mainly at date 3 [Fig. 6(B)].
SOD electrophoregrams indicated the presence of three
bands in both root and stem extracts, although the ®rst,
less mobile, band was hardly appreciable in stem samples
(Fig. 7). The ®rst two bands could be identi®ed as MnSODs since they were not inhibited either by KCN or
H2O2, whereas the third, faster moving, band was
characterized as a CuZn-SOD since it was negatively
a€ected by two inhibitors (not shown). The Mn-SOD
with higher mobility (Mn-SOD II), and the CuZn-SOD,
were the main SOD isoforms in roots and stems,
respectively (Fig. 7). In contrast with the enzyme activity
data, very little enhancement in the intensity of the SOD
electrophoretic bands was appreciated in samples from
infected plants as compared with non-infected controls.
As an unique possible exception, the level of CuZn-SOD
increased in infected roots of `WR315' compared with
non-infected controls at sampling date 2 [Fig. 7(A),
central panel, lane 4 versus 3]. This was in good
accordance with the signi®cant increase in SOD enzyme
activity found in the same sample [Fig. 2(E)].
Immunoblotting analysis of APX and SOD
In the same root extracts used for the spectrophotometric
and electrophoretic assays of enzyme activities, the
protein levels of SOD and APX isoforms were analysed
by Western blot by using polyclonal antibodies against
APX, CuZn-SOD and Mn-SOD (Fig. 8). In the case of
APX, an unique band of about 31 kD was observed
[Fig. 8(A)]. This band was stronger in infected roots of
`JG62' than in non-infected controls at all the three
sampling dates, whereas no inoculation-dependent
changes in APX band intensity were observed in roots
of `WR315' [Fig. 8(A)]. An unique band of 26 kD was
also revealed for Mn-SOD [Fig. 8(B)]. As a result of
C. GarcõÂa-Limones et al.
334
F I G . 8. Immunoblotting analysis for APX and SOD activities
from non-infected and Foc 5-infected roots of chickpea cvs.
JG62 and WR315. Protein levels were analysed at three times,
corresponding to seedlings without symptoms (sampling date 1),
at the onset of the ®rst symptoms (sampling date 2) and when
all plants had developed wilt symptoms (sampling date 3).
Transferred proteins from SDS±PAGE were probed with
polyclonal antibodies against cucumber APX (A), Equisetum
CuZn-SOD (B) and pea Mn-SOD (C). 1 and 2 are root samples
from non-infected and Foc 5-infected `JG62' plants. 3 and 4 are
root samples from non-infected and Foc 5-infected `WR315'
plants, respectively. In all cases, between 4 and 5 mg of protein
were loaded.
infection by Foc 5, this band showed an increase in
intensity at sampling date 1 in the compatible interaction,
and at date 2 in the incompatible one. The di€erences in
band intensities between infected and non-infected plants
were higher in `WR315' (incompatible reaction) than in
`JG62' (compatible reaction). Finally, in the case of
CuZn-SOD two bands of 15 and 17 kD, respectively,
were revealed. At all sampling dates, these bands were
more intense in `JG62' than in `WR315', and for both
cultivars reached their maximal intensities at sampling
date 2. As main infection-dependent changes, in `JG62'
(compatible interaction) the intensity of both bands
decreased at sampling dates 2 and 3, whereas in `WR315'
(incompatible interaction) the two bands increased in
intensity at sampling date 2 and the 17 kD band totally
disappeared at date 3 [ Fig. 8(C)].
DISCUSSION
In this work, we have studied various biochemical
parameters of oxidative metabolism during the interaction of the wilt-inducing race 5 of F. oxysporum f. sp.
ciceris with chickpea cvs. WR-315 (resistant) and JG-62
(susceptible). Although similar studies have been previously performed with a limited number of pathosystems, all of these studies refer to localized infections of
foliar tissues by obligate biotrophic or necrotrophic
pathogens for which incompatibility is associated with a
rapid development of HR [1, 19, 21, 23, 42, 56, 57].
Contrary to that, very little is known for plant±pathogen
interactions characterized by systemic infection, such as
fusarium wilt of chickpea, that are produced at root level
and that apparently do not develop such localized
resistant response [51].
The parameters included in our work are degree of lipid
peroxidation, activity levels of several antioxidant enzymes
(APX, CAT, GPX, GR and SOD) and levels of DAO
activity. It is worth noting that the above enzymes, besides
all being represented in the apoplast [56, 57], show
di€erent subcellular locations in such a manner that from
changes in their activities conclusions not only about the
production of AOS but also about the cellular compartment where it is occurring may be advanced. Finally, DAO
activity was included in our study because this enzyme
activity occurs at high levels in the apoplast of leguminous
plants, being the most abundant soluble protein of cell
walls from various of such plants including chickpea [20].
Also, DAO forms H2O2 which might participate in
resistance responses after pathogen attack [10].
In chickpea, the preferred site for infection of susceptible and resistant plants by F. oxysporum ciceris are the root
tissues close to the point of seed attachment. Fungal
hyphae colonize ®rst the root xylem and then the xylem
vessels of the stem [31, 51]. Therefore, the two plant tissues
sampled in our study represent in an sequential manner
the two scenarios where the plant±fungus interaction
leading to a compatible reaction is produced.
Results of the spectrophotometric assays indicate that
infection by F. oxysporum ciceris led to substantial changes in
the antioxidant status of chickpea, although there were
clear di€erences between compatible and incompatible
interactions as well as between root and stem plant tissues
in the responses produced. One can distinguish between
responses associated to both compatible and incompatible
interactions from those speci®c for either the compatible or
the incompatible interaction. Thus, the common responses
were the increases in the degree of lipid peroxidation and
CAT and SOD antioxidant enzyme activities that resulted
from infection by the pathogen. The induction of CAT and
SOD seems point out to an enhanced O2 and H2O2
production, being worth noting, with respect to the
putative H2O2 production, the sole induction of one of
the two plant detoxifying mechanisms, that involving
CAT, which is precisely the more localized one considering
the subcellular compartmentation of them. Thus, whereas
CAT is primarily con®ned to peroxisomes, APX, which
forms part of the other main plant H2O2 detoxifying
mechanism [5], has been found in practically all plant cell
compartments, including chloroplasts, microbodies, cytosol and mitochondria [30]. Characteristically however,
responses common to both interactions were produced
earlier in the incompatible than in the compatible
interaction. Therefore, if the above responses are important for resistance they may have occurred too late in the
susceptible interaction to a€ord protection.
Induction of an antioxidant enzyme system and other oxidative stress markers
Responses speci®cally linked to the compatible interaction were the induction of APX, GR and, less
importantly, GPX. The former two enzyme activities
clearly relate to the ascorbate±glutathione cycle [5], the
most general mechanism of H2O2 detoxi®cation in plants.
Since the pathogen enters into the root and stem xylem of
susceptible chickpeas but not that of resistant plants [51],
one might speculate that the activation of the ascorbate±
glutathione cycle is a response to the H2O2 production in
the xylem parenchyma due to the presence of the
pathogen in this tissue. In fact, the down-regulation of
APX has been found associated with the expression of
resistance, rather than with that of susceptibility, in some
instances [19, 57]. Two other possible causes for the above
responses speci®cally linked to the compatible interaction
would be that they were produced by either: (i) the
pathogen, as a response aimed to cope with the putative
plant oxidative burst; or (ii) the plant, as a general
response to water stress caused by development of the
wilting syndrome. With respect to the ®rst possibility,
although is likely that pathogens exposed to sublethal
doses of AOS may increase their antioxidant defences, this
oxidative stress adaptation and its possible role in
virulence has been little studied for fungal plant
pathogens [41]. However, as no di€erences in APX and
GPX isoenzyme patterns were found in infected versus
non-infected plants, the alternative (i) would require the
very unlikely condition that the two above enzymes would
show the same electrophoretic multiplicity and mobility
irrespective of being of plant or fungal origin. With respect
to possibility (ii), the e€ect of water de®cit mainly lead to
a situation of excessive excitation energy similar to that
associated with high light in leaves [49]. Thus, it might be
expected that in such a situation the antioxidant defences
would be induced in green rather than in root tissues.
Lastly, only one response, the early induction of DAO
activity in stems, could be speci®cally associated to the
incompatible interaction. As this enzyme activity forms
H2O2 in the apoplast, this result is consistent with a
possible role of AOS generation in the resistance of
chickpea to fusarium wilt. However, more evidence is
needed for considering both the relevance of this defence
mechanism and the implication of DAO in it.
Not all the above responses to pathogen infection in
compatible and incompatible interactions were similarly
expressed in both root and stem tissues. The speci®c
induction of DAO activity in stem tissues at sampling
date 1 was the only signi®cant change produced as a
consequence of infection of the resistant `WR315'.
In contrast, in the susceptible `JG62'±Foc 5 interaction,
there were no di€erences between stems and roots, which
showed inductions as result of infection in either the
speci®c responses of the compatible interaction (APX,
GR and GPX activities) as well as in responses common
to both interactions (lipid peroxidation degree and CAT
335
and SOD activities). This lack of induction of antioxidant
enzymes in stems of the resistant plants might relate to
lack of colonization of these tissues by the pathogen,
which would relate oxidative stress and pathogen spread.
On the other hand, it might simply denote a less ecient
AOS scavenging mechanism and, hence, an increased
AOS level in the incompatible interaction, which would
relate oxidative stress and resistance. As with other plant
defence responses, the time course of AOS production in a
given plant±pathogen interaction may probably determine its role as either a resistance reaction or just a late
response to pathogen spread.
Summarizing, results from this present work show the
induction of the antioxidant enzyme system and other
oxidative stress markers during fusarium wilt of chickpea,
suggesting that changes in oxidative metabolism may be a
quite general plant defence response not only restricted to
foliar pathogens causing resistant reaction through HR
and necrotic processes. Our results also suggest that
increased levels of AOS, built up by either enhanced
production and decreased scavenging potential, may
contribute to the resistance reaction in chickpea to
fusarium wilt. Studies now in progress about direct AOS
estimation and induction of AOS-forming enzymes,
induction of non-enzyme antioxidants and compartmentation of antioxidant responses at the apoplast level, will
probably provide more conclusive insights about the
production of an oxidative burst and related responses and
their role in the pathogenesis of fusarium wilt of chickpea.
Immunoblotting analyses were performed by MC G-L
during a short stay made at Dr LA del RõÂ o's laboratory
(EstacioÂn Experimental del Zaidin, CSIC, Granada,
Spain) where she used antibodies available in that
laboratory. This work was supported by DGESIC
(Spain), Project PB97-0444, and Junta de AndalucõÂ a
(Spain), PAI Research Groups AGR 136 and AGR 164.
MC G-L is recipient of a predoctoral fellowship from the
Ministerio de EducacioÂn y Ciencia (Spain).
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